干旱胁迫下黄檗幼苗cDNA消减文库的构建和分析

作者：王韵壹、李英、熊慧卿

生物工程学报 Chin J Biotech 2008, February 25; 24(2): 198-202

journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061

Received: May 16, 2007; Accepted: June 12, 2007

Supported by: the State Key Basic Research and Development Plan of China (No. G19990160).

Corresponding author: Yuangang Zu. Tel: +86-451-82191517; E-mail: zygorl@vip.hl.cn

国家重点基础研究发展规划项目(No. G19990160)。

研究报告

干旱胁迫下黄檗幼苗cDNA消减文库的构建和分析

王慧梅, 王延兵, 祖元刚, 孙莲慧

东北林业大学森林植物生态学教育部重点实验室, 哈尔滨 150040

摘要: 以干旱胁迫下的黄檗幼苗cDNA 为tester, 正常生长的黄檗幼苗cDNA为driver, 利用抑制性消减杂交技术(suppression subtractive hybridization, SSH)构建了干旱胁迫下黄檗幼苗的消减文库并对其进行了EST序列分析。从消减文库中随机挑取20个阳性克隆, 提取质粒进行酶切和PCR鉴定, 显示文库克隆的重组率大于95%, 插入片段大小大部分集中在300~800bp之间。随机挑取816个克隆进行测序, 得到265个基因。将其进行同源性分析, 划分为16类。获得了热激蛋白70、脱水响应蛋白(RD22)、通用胁迫蛋白、金属硫蛋白(MTII), 晚期胚胎丰富蛋白(LEA14)等44种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。本研究为抗逆基因克隆和系统研究干旱胁迫下黄檗基因的表达奠定了重要的理论基础。

关键词: 干旱胁迫, 黄檗, 抑制性消减杂交, EST序列分析

Construction and Analysis of Subtractive cDNA Library of Phellodendron amurense Under Drought Stress

Huimei Wang, Yanbing Wang, Yuangang Zu, and Lianhui Sun

Key Laboratory of Forestry Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China

Abstract: With cDNA from Phellodendron amurense seedlings treated with drought stress as tester and cDNA from this plant in normal growth as driver, we construct cDNA subtracted library using suppression subtractive hybridization (SSH). In the library, the rate of recombination was 95%, the size of inserts was 300~800 bp. Two hundred and sixty-five new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after nucleotide Blast and BlastX homological analysis against NT, NR, SWISSPROT, KEGG database. Forty-four drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothionein II, late embryogenesis abundant protein, were obtained, which made 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and antioxidant enzyme. The research had established a basis for cloning stress resistance genes and further studying genes expression in P. amurense seedlings under drought stress.

Keywords: drought stress, Phellodendron amurense, suppression subtractive hybridization, expressed sequence tags

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